With a case fatality rate higher than the 1918 Spanish Flu pandemic, H5N1 highly pathogenic avian influenza represents a threat to global public health. Efforts to identify locations with the greatest potential for pandemic emergence, as well as how the virus is spreading, may help minimize this threat. First detected in Egypt in 2006, H5N1 viruses have resulted in the deaths of millions of birds in both commercial and backyard poultry flocks, and more than 350 human infections, the most of any country, have been confirmed. Human outbreaks have been so far constrained by poor viral adaptation to non-avian hosts. There are two evolutionary mechanisms by which the H5N1 avian influenza virus could acquire pandemic potential: 1) via reassortment as a result of coinfection with another subtype (such as low pathogenic avian influenza H9N2); and/or 2) via antigenic drift and the accumulation of randomly occurring genetic changes found to improve viral fitness, herein called key substitutions (KS). Both mechanisms were investigated using geospatial methods including ecological niche modeling and hot spot analyses to predict locations with elevated potential for pandemic emergence. Using ecological niche modeling environmental, behavioral, and population characteristics of H5N1 and H9N2 niches within Egypt were identified, with niches differing markedly by subtype. Niche estimates were combined using raster overlay to estimate co-infection potential, with known occurrences used for validation. Co-infection was successfully predicted with high accuracy (area under the receiver operating characteristic (ROC) curve (AUC) 0.991). 41 distinct KS in H5N1 were detected in Egyptian isolates, including 17 not previously reported in Egypt. Phenotypic consequences of detected KS were varied, but the majority have been implicated in improving mammalian host adaptation and increasing virulence. Statistically significant spatial clustering of high KS rates was detected in the northwestern portion of the Nile River delta in the governorates of Alexandria and Beheira. To investigate how the virus spreads between poultry farms, landscape genetics techniques were employed. Viral genetic sequences were evaluated using phylogenetics to determine viral relatedness between samples, then distance models representing competing diffusion mechanisms were created using road networks and a least-cost path model designed to approximate wild waterbird travel using niche modeling and circuit theory. Spatial correlations were evaluated using Mantel tests, Mantel correlograms, and multiple regression of distance matrices within causal modeling and relative support frameworks. Samples from backyard farms were most strongly correlated with least cost path distances, implicating wild bird diffusion, while samples from commercial farms were most strongly correlated with road network distances, implicating human-mediated diffusion. Results were largely consistent across gene segments. Identifying areas at risk of co-infection can help target spaces for increased surveillance. Similarly, detecting spatial hot spots of KS highlight areas of concern for pandemic emergence from antigenic drift. Demonstration of different diffusion mechanisms by farm type should inform both surveillance and biosecurity practices. Knowledge of where to focus intervention efforts, both spatially and strategically, allows limited public health resources to be targeted most effectively. By detecting where in the country pandemic influenza is likely to emerge and identifying how the virus is spreading between farms, this work contributes to efforts to predict and prevent the next influenza pandemic.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

This dissertation examines issues related to avian influenza (AI) disease. This is done via three essays that individually examine: (1) the impacts of climate change on the probability and expected numbers of AI outbreaks and associated economic loss; (2) the effects that media coverage of AI outbreaks has on meat demand in the United States, and (3) the potential effectiveness of AI mitigation strategies on poultry production and welfare under a simulated AI outbreak in United States. The climate change and spread of AI outbreaks study finds that the probability and expected number of AI outbreaks increases as climate change proceeds. Particularly, past climate change has contributed to the current spread of AI disease by 11% and the future climate change will increase this spread by another 12%. Moreover, the underreporting probability of AI outbreaks is also examined and results show that the underreporting probability is much higher in countries with lower gross domestic production level, larger export of poultry products and more numbers of AI confirmed human deaths. Therefore, disease prevention and control plans should focus on these economically poor and climatically changed regions. AI outbreak information has significant effects on meat demand in the United States. In particular, impacts of overseas AI human deaths on meat demand equal 0.02% for beef, -0.005% for pork, and -0.01% for chicken for sample when there was no AI occurred in the United States, while it has smaller impacts on meat expenditure when using the whole sample. In addition, human deaths due to AI disease will increase beef demand and decrease that for pork and chicken. However, AI media coverage in short-run has insignificant effect on meat demand, which suggests that consumers are more cautious when cases occur within the United States as opposed to international cases. In the study on the effects and welfare implications of AI mitigation strategies, results find that vaccination strategy is welfare decreasing under most cases of demand shocks but is desirable in some regions when both domestic and excess demand decrease. Under the assumption of one AI outbreak in the United States, the associated mitigation costs because of past climate change are relatively small.

Although avian influenza virus (AIV) infections in domestic poultry are uncommon, transmission of avian influenza from wild waterfowl reservoirs does occur. Depopulation of the infected flock is the typical response to AIV outbreaks in domestic chicken production, causing a loss in profits and accumulation of unexpected expenses. Because it is impossible to know which of many virus subtypes will cause an outbreak, it is not feasible for the U.S. to stockpile vaccines against all possible avian influenza threats. Currently, the U.S. does not routinely vaccinate chickens against influenza due to the inability to differentiate infected from vaccinated animals (DIVA), which would place limitations on its trade markets. A Sindbis virus vector expressing the PR8 influenza strain's M2e peptide was developed as a potential universal DIVA vaccine. M2e is a conserved peptide amongst influenza A viruses; M2e-specific antibodies induce antibody-dependent cytotoxicity or phagocytosis of infected cells, reducing production and shedding of AIV during infection. In this study, chickens were vaccinated at one-month-of-age with parental (E2S1) or recombinant Sindbis viruses expressing the PR8 M2e peptide (E2S1-M2e) by subcutaneous or intranasal routes at high (106 pfu) or low (103 pfu) dosages. Chickens were boosted at 2-weeks post-initial vaccination using the same virus, route, and dosage, then challenged with low pathogenic H5N3 AIV at 0.2 mL of 106/mL EID50 2-weeks post-boost. Serum samples were collected at 1-week and 2-weeks post-vaccination, 2-weeks post-boost, and 2-weeks post-challenge and screened for PR8 M2e-specific IgY antibody production by ELISA. Both high and low dose subcutaneously, as well as high dose intranasally vaccinated E2S1-M2e groups produced significantly higher levels of PR8 M2e-specific IgY antibodies as early as 1-week post-vaccination, while the uninoculated control and E2S1 groups remained negative for all pre-challenge time points. M2e-specific IgY antibodies capable of binding the challenge H5N3 M2e peptide were detected in groups with existing vaccine-induced M2e-specific antibodies pre-challenge, suggesting antibody M2e cross-reactivity. After challenge, all groups developed M2e-specific IgY antibodies and high HI titers, verifying successful AIV infection during challenge and production of hemagglutinin-specific antibodies. Viral shedding titers 4-days post-challenge were used to measure vaccine efficacy and were similar amongst all groups. Microneutralization assay results confirmed that post-boost serum samples, containing only M2e-specific antibodies, were unable to neutralize AIV in vitro. Although the E2S1-M2e vaccine was capable of producing high levels of M2e-specific IgY antibodies when inoculated subcutaneously, these antibodies were not able to reduce viral shedding and therefore did not protect chickens from AIV.

The avian influenza virus can be transmitted from sick birds to other birds by fecal and oral secretion. Therefore, when birds with avian influenza virus enter domestic bird farms and make contacts, it is possible to carry the virus in and transmit it to domestic birds. Since the outbreak of the avian influenza in Taiwan, most bird farms had set up the screen. Yet, the actual effectiveness of screen coverage rate has not been researched. This study will discuss further regarding the results of different screen coverage rate in preventing wild birds¡¦ entering to poultry farms in Kaohsiung area. Field observations were carried out at the wetlands in Kaohsiung county, including Old Railroad Bridge artificial wetland, Fongshan reservoir and 20 poultry farms which were less than five kilometers away from wetlands from September 2008 to October 2009. Chicken and duck farms were analyzed respectively, and the screen coverage rate was categorized into full, partial and no coverage, respectively. The aims of the study were to investigate the effect of screen coverage on the entrance of wild birds to the farms, and found out the potential bridge species near poultry farms. By observing different levels of screen coverage in poultry farms, migratory birds¡¦ activity, and the contact situation between wild and domestic birds, we can analyze the contact level of high risk species and speculate possible transmission path of the avian influenza, and understand the effectiveness of the screens. Also, showing the correlation between the density of domestic poultry and the number of wild bird enterance. The result showed, in terms of screen with different coverage rates, the number of bird species within the duck farms had significant different in full and no coverage. The number of total birds within the duck and chicken farms with full coverage was significant less than partial coverage. Both in chicken and farms, screen had a great effectively in preventing small sized birds entering, yet not on big sized birds. The density of domestic poultry and the number of wild bird enterance showed negative correlation only in chicken farms with partial coverage. In terms of the wild land birds entering poultry farms, Passeer montanus is the largest species, followed by Hirundo rustica and Streptopelia tranquebarica. As for aquatic birds, it is mainly of Ardeidae, Scolopacidae, Charadriidae and Rallidae. Among all, Passeer montanus, Hirundo rustica, Streptopelia tranquebarica, and Bubulcus ibis are not migratory birds and highly pathogenic avian influenza virus species which move between poultry farms and wetlands. Therefore, they were considered bridge species. Due to the bridge species makes direct contacts with poultry, it might be the spreader of the virus. Decreasing the possibility of wild birds¡¦ contact with poultry is an important mechanism for avian influenza control. Also, based on the model of European Food Safety Authority (ESFA), risk assessments will be conducted on Kaohsiung County wetland¡¦s migratory birds¡¦ highly pathogenic avian influenza virus H5N1 transmission to poultry farms.

We collected 6,823 cloacal swabs over four years (2005–2006: 1,460; 2006– 2007: 2,171; 2007–2008: 2,424; and 2008–2009: 768) from 30 potential avian host species. Most samples (88.3 percent) were from dabbling ducks (genus Anas), while diving ducks (genus Aythya) accounted for 5.0 percent, and geese (genera Anser, Chen, and Branta) 3.0 percent of the samples tested. Waterfowl (Anatidae) comprised 98.7 percent of samples, with 1.8 percent from non-migratory dabbling ducks (genus Anas). All samples were screened for avian influenza virus (AIV) by AIV-matrix real-time RT-PCR (rRT-PCR); all rRT-PCR positive samples (541) were processed for virus isolation as well as 4,473 rRT-PCR negative samples. Differences were observed in apparent prevalence estimates over the four years between virus isolation (0.5, 1.3, 3.9, and 0.7 percent) and rRT-PCR (5.9, 6.5, 11.2, and 5.5 percent). We isolated 138 AIVs, of which two were obtained from rRT-PCR negative samples. Unlike previous reports of seasonal variation in AIV prevalence, we documented differences in prevalence estimates among months using rRT-PCR only during 2008–2009 and by virus isolation only during 2006–2007 and 2007–2008. Several of the AIV subtypes we identified are common in North America (e.g., H3, H4, and H6); H3N8 and H4N6 were the most common subtype combinations isolated. Similar to most surveillance studies, we found no significant difference in AIV infection based on host sex, but did find that juveniles were more likely to be positive for AIV than adults. We also documented that dabbling ducks were more likely to be positive for AIV than diving ducks, although not all dabbling ducks are equally likely to be positive. Molecular sequence analysis revealed no insertions of multiple basic amino acids at the cleavage site, which supported the identification of low pathogenic AIV. Phylogenetic anlyses performed on H5, H6, H7, N1, N2, N3, and N4 subtypes sequenced indicated similarity to other North American isolates with the exception of seven H6 which were more similar in amino acid translation to an isolate from Japan. In sum, this is the first multiyear study of avian influenza viruses on waterfowl wintering grounds of the Central Flyway, a historically understudied area of North America.

Avian Influenza (AI) has made hundreds of people dead in Europe. The hospital system of Taiwan, standing in the first line of prevention and treatment, has to effectively employ the experience of SARS in order to tackle the new serious challenge of AI. Though Taiwan is not yet an epidemic area of AI, it is important for us to be well prepared to confront it. Given that prevention is more important than treatment. It is essential that we try our best to build up an efficiently preventive system for AI. This study aims to investigate what is most important and critical in the preventive system. More specifically, what is a priority to do in such a preventive system, especially in a limited condition of budgets and human resources? According to the five stages of crisis management proposed by Mitroff (1988) (see page 35), the current study focuses mainly on operations in the second stage, preparation and prevention of crisis. In terms of analytic hierarchy process (AHP), it designs a questionnaire to systematically interview 12 doctors. Since four of them are outliers when the consistency test is considered, results are based primarily on the opinions of the remaining 8 doctors. The results are summarized as follows. 1. Before the crisis comes, both ¡§the training of doctors and nurses¡¨ (weighted value = 0.3195) and ¡§the regularization of preventive process¡¨ (weighted value = 0.2978) are most important. 2. The regularization of preventive process (weighted value = 0.3066) is most economic in the limited condition of budgets that is available to the preventive operations. Accordingly, it is a best way to regularize the individual operations and spread the concept of the regularization. 3. The training of doctors and nurses (weighted value = 0.3513) is most important in the limited condition of human resources, that recently appears gradual reduction among hospitals. That is, by means of rehearsal of related operations and of training courses, the crew in a hospital can have an understanding of AI spreading in a short period of time. 4. In consideration of hard equipments, the doctors to be questioned hold similar opinions, as mentioned above. Apparently, the mastery of various preventive operations in the preventive system plays a critical role in that whether the AI crisis will continue to develop. 5. Only in consideration of the effects of prevention, the training of doctors and nurses is also most important. In conclusion, the experts all think that the training of doctors and nurses is a priority, regardless of in what conditions. The findings of the study hope to provide references for other hospitals by manifesting the order of the importance of different preventive operations in the preventive system.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Continually reported human cases of highly pathogenic avian influenza (HPAI) H5N1 virus infection create heightened threat to public health, due to the disease severity and high lethality. Acute respiratory syndrome (ARDS) has been found to be the most severe form of acute lung injury caused by H5N1 virus infection. Studies have highlighted that the unusually high virulence of H5N1 virus infection is associated with the cytokine dysregulation and enhanced viral replication in the host. In reference to the past experience during Spanish 1918 influenza pandemic and SARS, it is crucial that a novel therapeutic target is explored and employed in time for the effective control of emerging diseases. The pandemic potential of H5N1 influenza virus urges well preparedness not only in terms of containment measures, but also in the treatment aspect of the severe human H5N1 disease. To date, therapeutic approaches are limited to the use of vaccine, antiviral drugs and corticosteroids. It has been suggested that commercially available antiviral drugs are prone to induce resistance mutations; and are effective in the protection against influenza virus infection only if administered during the early course of disease development. Moreover, vaccine development does not grant a promising therapeutic strategy at the time of a pandemic as it takes time for the development and distribution of safe and reliable vaccine. In attempts to search for a novel adjunctive therapy in addition to currently available agents, indirubin-3’-oxime (IDO) and indirubin derivative, E804 have been tested to show the effect in cytokine suppression and antiviral activity against H5N1 influenza virus infection in vitro. These compounds have been extracted and purified from a natural herb called Isatis tinctoria which is frequently used for herbal remedy in treating respiratory symptoms in traditional Chinese medicine. In this study, it was demonstrated that IDO and E804 treatment in H5N1 influenza virus infected human alveolar epithelial cells effectively inhibit the proinflammatory cytokine induction and viral replication. This physiologically relevant in vitro alveolar epithelial cell model and the efficacy of IDO and E804 provide new insights to the development of new treatment option for severe human H5N1 disease. / published_or_final_version / Microbiology / Master / Master of Medical Sciences